Pathogenic Yersinia species use a virulence plasmid encoded type III secretion system to transport effector proteins (Yops) via a needle structure into the cytoplasm of immune cells, thereby preventing phagocytic clearance of the invading pathogen. Yersinia type III secretion machines are constructed from 26 Ysc proteins, forming a needle-like structure and creating a conduit through which effectors are injected into host cells. Type III machines assume rotational symmetry and several of their components share significant structural homology with the basal body of flagella and with type III machines of other bacteria. Assembly of type III machines requires the secretion of early substrates, namely YscF, which polymerizes into the hollow needle structure, as well as YscP and YopR. Once needle formation is completed, middle substrates, known as translocators, are deposited at the needle tip (LcrV) or in the plasma membrane of host cells (YopB and YopD) to generate the translocation pore. Type III machines encounter a drop in perceived calcium levels in the cytosol of host cells, which is thought to impact the structure of the needle and trigger the transport of late substrates, for example YopE effector, a GTPase activating protein (GAP). Our experimental inquiries are concerned with the events that enable Yersinia to select substrates for organized assembly or secretion by type III machines. Using translational hybrids of early, middle and late substrates with reporter proteins that do or do not pass through the secretion machine, we observed that entry into the pathway requires at least two signals. N-terminal signals initiate all substrates into the type III pathway and these are likely decoded by features of mRNAs that encode proteins destined for travel. Secondary signals are positioned downstream and establish order, i.e. they determine the sequence of assembly and transport events. One of these secondary signals, a stem-loop structure in the 3' coding sequence of yopR mRNA, promotes selection of its product as an early substrate, presumably by programming ribosomes for secretion. Switches in substrate recognition occur at the level of each needle complex and are associated with autoproteolytic cleavage of YscU, the switch protein, and its variable interaction with other machine components, including the type III ATPase complex (YscN-YscK-YscL-YscQ) and YscO. Goal of this proposal is to understand the mechanisms whereby type III machines select substrates for ordered secretion reactions. Further, utilization of newly developed microscopy technologies can now reveal the mechanisms whereby Yersinia select primary host cells for type III injection. Detailed appreciation of how bacterial pathogens use type III machines will enable therapeutic intervention and prevention of many different infectious diseases.